The present invention relates to a method of screening compounds capable of reducing cellular damage associated with an ischemic condition, including stroke, glaucoma and other neurodegenerative diseases, as well as myocardial infarction, and to methods of treating patients who are susceptible to or who exhibit ischemia-related cellular damage.
Ischemic injury to cells and tissues occurs as a result of a number of insults that result in decreased perfusion with oxygenated blood, e.g., cerebral ischemia (xe2x80x9cstrokexe2x80x9d), myocardial infarction and reperfusion injury (Walton, et al., Neuroreport 8(18):3871-3875 (1997); MacManus, et al., Neurosci. Lett. 164:389-92 (1993)). Two distinct patterns of pathologic cell death are generally associated with cellular ischemia: necrosis and apoptotic cell death. As described in greater detail in Section II, below, each of these types of cellular death are characterized by distinct, recognizable morphological and biochemical characteristics. Numerous diseases have been associated with faulty regulation of apoptosis including, e.g., neurodegenerative conditions, AIDS and vascular disease. [Allen, et al., Cell Mol. Life Sci. 54(5):427-445 (1998)].
Ischemic damage to the central nervous system (CNS) may result from either global or focal ischemic conditions. Global ischemia occurs under conditions in which blood flow to the entire brain ceases for a period of time, such as may result from cardiac arrest. Focal ischemia occurs under conditions in which a portion of the brain is deprived of its normal blood supply, such as may result from thromboembolytic occlusion of a cerebral vessel, traumatic head injury, edema, and brain tumors. Both global and focal ischemic conditions have the potential for producing widespread neuronal damage, even if the ischemic condition is transient.
Animal models have been established that mimic the symptoms of both global and focal cerebral ischemia, most notably, the gerbil model of global ischemia produced by transient occlusion of carotid arteries of the neck. [Kirino, Brain Res. 239:57-69 (1982)], the rat four-vessel occlusion model for ischemia [Pulsinelli, et al., Stroke 10:267-272 (1979)], the MCAO microfilament of focal ischemia [Tamura, et al., Journal Cereb. Blood Flow Metab. 1:53 (1981)], and a rat model for glaucoma [Isenmann, et al., Eur. J. Neurosci. 9:1763-1772 (1997)]. Although animal models are important sources of information as to which candidate therapeutics are likely to be efficacious in mammals, they are costly, time consuming and not amenable to screening large numbers of compounds.
There exists a need for an in vitro screening system effective to discriminate between the large number of potential therapeutic agents that are available for treatment of ischemia- and apoptosis-related disorders. The present invention provides reliable, reproducible predictor assays that are adaptable to screening large numbers of test compounds in parallel. Such in vitro assays facilitate selection of candidate compounds, which can then be tested in relevant animal models to determine if they are suitable for administration to human patients. The present invention also provides compounds and methods of treatment based on performance of candidate compounds in such in vitro assays.
The present invention is directed to the discovery of in vitro methods for selection of candidate therapeutic agents for in vivo treatment of disorders having as their underlying etiology, ischemia-related cellular damage or death. Such disorders include, but are not limited to ischemia, glaucoma and other neurodegenerative diseases, as well as cardiac injury associated with myocardial infarction. While such disorders are usually characterized by apoptotic cell death, apoptosis or necrosis may or may not be involved.
The present invention is also directed to the use of such in vitro methods for determining a therapeutically effective amount of a given candidate therapeutic agent for the in vivo treatment of such disorders in a subject.
The present invention is based, in part, on the discovery of a method for selecting compounds which are candidates for treatment of ischemia-related cellular damage. The invention includes a method for evaluating the relative efficacy of such compounds based on decreased cell death in oxygen/glucose-deprived excitable cells in culture, exemplified by neuronal cells, such as retinal ganglion cells (xe2x80x9cRGCsxe2x80x9d) or myocardial cells, such as myocytes, in in vitro primary cultures. In a preferred embodiment, such cells are prepared in culture to be at least 80% and preferably at least 90%, and more preferably 99% homogeneous with respect to other cells in the culture or cell population employed in the assay.
The present invention is also based on the discovery of a method for selecting test compounds that are candidate cellular protective agents for treatment of myocardial infarction, glaucoma and other neurodegenerative diseases. In this aspect, the invention includes a method for evaluating the relative efficacy of such test compounds based on decreased cell death in growth factor-deprived or oxygen/glucose and growth factor-deprived retinal ganglion cells in vitro.
In the assays of the present invention, cell death may be related to apoptosis or necrosis. Accordingly, the relative efficacy of test compounds for treatment of ischemia-related neuronal cell damage, including glaucoma and other neurodegenerative diseases may be evaluated in the retinal ganglion cell models of the present invention using endpoints indicative of apoptotic and/or necrotic cell death. Likewise, the relative efficacy of such test compounds for treatment of myocardial cell damage, such as caused by myocardial infarction, may be evaluated in myocyte cell models of the present invention.
In a related aspect, the present invention provides a method for reducing cellular damage related to an ischemic condition by administering to a subject, a therapeutically effective amount of one or more test compounds as determined by the relative efficacy of the one or more test compounds in reducing cell death due to the ischemic condition in an in vitro assay of oxygen/glucose-deprived excitable cells, as exemplified by retinal ganglion cells or myocytes. In the present invention, the in vitro cell death of oxygen/glucose-deprived retinal ganglion cells may occur by an apoptotic or necrotic mechanism.
In another aspect, the present invention provides a method for reducing cellular damage related to myocardial infarction, glaucoma or another neurodegenerative disease by administering to a subject, a therapeutically effective amount of a test compound as determined by the relative efficacy of the test compound in reducing cell death due to the ischemic condition in an in vitro assay of growth factor or oxygen/glucose and growth factor-deprived retinal ganglion cells. In the present invention, the in vitro cell death of growth factor or oxygen/glucose and growth factor-deprived retinal ganglion cells generally occurs by an apoptotic or necrotic mechanism.
These and other objects and features of the invention will become more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying drawings.